Elastin is a protein presented in the elastic fiber of the arteris, dermis etc. of vertebrates. Elastin has a large amount of special bridging structure such as desmosine and lysinonorleucin. The amino acid composition thereof is very special, with 50-60% of alanine and glycine. It is water-insoluble and cannot be easily hydrolyzed by ordinary proteases (Foster (1982) Methods Enzymol., 82, 559-570; and Paz et al., (1982) Methods Enzymol., 82, 571-587). Proteases capable of hydrolyzing elastin are specifically called as elastases, including porcine pancreatic elastase (Ardelt et al. (1970) Biochim, Biophys. Acta, 341, 318-326) and the proteases secreted by microorganisms such as Pseudomonas aerugionsa (Morihara et al. (1965) J. Biol. Chem., 240, 3295-3304), Streptomyces griseus (Gertler et al (1971) Eur. J. Biochem., 240, 3295-3304) and Flavobacterium sp. (Ozaki et al. (1975) J. Biochem., 77, 171-180). It is generally considered that elastase is capable of hydrolyzing elastin as having (a) stronger binding affinity for elastin, and (b) stronger substrate specificity for alanine and glycine (Stone et al. (1982) Methods Enzymol., 82, 588-605).
Bacillus sp. YaB is an aerobic gram positive bacterium. It is a rod-shaped bacillus capable of producing spores and it may form a yellow colony on the culture medium. The cell wall thereof contains D,L-diaminopimelic acid. The growth of the bacterium is better in the medium at pH 10 than under neutral environment. Bacillus sp. YaB has been deposited in the Food Industry Research and Development Institute (FIRDI), Hsin-Chu, Taiwan, under Accession No. CCRC 11751.
The enzyme of the invention, subtilisin YaB (also referred to hereinafter as protease YaB), is originally named as alkaline elastase YaB. It is an extracellular protease produced by alkalophilic Bacillus YaB. The enzyme has a molecular weight of about 27,000 and an isoelectric point of 10.6. The optimal reaction pH thereof is 11.75. It is highly hydrolytic for elastin and is therefore originally named as elastase YaB. Moreover, it is also substantially more hydrolytic for collagen than other subtilisin proteases. See, Tsai et al. (1983) Biochem. Int., 7, 577-583; Tsai et al. (1984) Biochem. Int., 8, 283-288; Tsai et al. (1986) Biochim. Biophys. Acta, 883, 439-447; Tsai et al. (1988a) Appl. Environ. Microbiol., 54, 3156-3161; and Tsai et al. (1988b) J. Biochem., 104, 46-420.
The high hydrolyzing activity of the enzyme for elastin can be demonstrated by its binding affinity to elastin and its enzyme-substrate specificity. Within the pH range lower than its isoelectric point, the enzyme has a very high binding affinity to elastin. At pH 7.0, after mixing 50 mg of the enzyme with 5 mg elastin, more than 60% of the protease YaB are bound to the elastin. Under the same conditions, only 60% of subtilisin BPN' and 5% of chymotrypsin are bonded. See, Tsai et al. (1986) Biochim. Biophys. Acta, 883, 439-447. With respect to substrate specificity, it is for large aromatic amino acids such as tyrosine and phenylalanine for typical subtilisins. However, the subject enzyme of the invention has the substrate specificity for small amino acids such as alanine and glycine, which meets to the characteristics of elastase. See, Tsai et al. (1984) Biochem. Int., 8, 283-288.
The gene of the above enzyme has been cloned in E. coli and can be expressed in Bacillus. The nucleotide sequence of the gene has also been determined. See, Kaneko et al. (1989) J Bacteriol., 171, 5232-5236. The primary structure of the enzyme deduced from the nucleotide sequence shows about 60% homology with other subtilisins, including subtilisin BPN', Carlsberg and the like. Therefore, the enzyme should be a member of the subtilisin family and its name should be changed to subtilisin YaB. This enzyme has distinctive characteristics among subtilisins. In addition to the aforementioned substrate specificity for small amino acids, the optimal reaction pH of the enzyme is 11.75, while the optimal reaction pHs of typical subtilisins are all between about 9.0 to 10.5.
With respect to the applications, the enzyme has the full potential to be developed to a meat tenderizing enzyme (Takagi et al. (1992) J. Agric. Food Chem., 40, 2364-2368). Typical meats such as beef, pork and chicken all have connective tissues containing large amounts of hard proteins such as elastin and collagen. Commercially available meat tenderizers contain plant proteases such as papain and bromelain for hydrolyzing those hard proteins so as to increase the tenderness of meats. However, the substrate specificities of these protease are too low. In addition to elastin and collagen, myofibrillar proteins such as actin and myosin may also be hydrolyzed. Therefore, the taste of meats will be deteriorated. The enzyme of the invention has the substrate specificity that is substantially higher than those of these proteases such as papain. When used in the treatment of meats, the enzyme can hydrolyze the elastin and collagen contained in the meat while only exert little hydrolyzing activity on actin and myosin. The treatment of meat with the enzyme of the invention result in good taste.
However, because the substrate specificity of the currently available protease YaB is still not sufficiently strict, it is difficult to control the conditions for the treatment of meat. If the administration concentrate of the enzyme is too high in parts of the meat, the taste of meat may still be deteriorated. Therefore, it is still desired in the art to obtain a protease with even higher substrate specificity for hard proteins such as elastin and collagen, in particular those with little, even no activity for myofibrillar proteins such as actin and myosin.
None of the above background references has disclosed or suggested that the substrate specificity of subtilisin YaB can be enhanced and/or altered by protein engineering techniques, so as to enhance the efficacies of the enzyme in its known applications and/or to develop other new applications.